Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid

Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid Ling Zhu, Jiang-Yan Yu, Qing-Fang Xu, Xu Bai, Xiu Gao, Li-Fang Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4842597/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Archives of Microbiology → Version 1 posted 13 You are reading this latest preprint version Abstract Pyracantha fortuneana ( P. fortuneana ) fruit, as a dual-purpose plant resource, is rich in nutrients; however, studies on the isolation and identification of yeast strains from P. fortuneana fruit and their brewing characteristics are scarce. To screen for high-quality yeast strains specifically for P. fortuneana wine from the fermentation liquid, this study employed traditional pure culture methods using WL nutrient agar for morphological identification of the isolated strains. Molecular biological identification of the yeasts was performed using molecular biology techniques. The brewing characteristics of the yeast strains were analyzed based on growth characteristics, sugar tolerance, alcohol tolerance, SO 2 tolerance, and acid tolerance, providing potential quality yeast resources for P. fortuneana wine production. The results indicated that a total of 12 yeast strains were isolated and purified. After identification, they were classified as Pichia kudriavzevii and Saccharomyces cerevisiae . Strain HJ-2 was able to grow normally under the conditions of 40°C, 15% alcohol by volume, 360 mg/L SO 2 mass concentration, pH 3.2, 400 g/L sucrose mass concentration, and 250 g/L glucose mass concentration. Strain HJ-6 exhibited normal growth under conditions of 40°C, 3% alcohol by volume, 360 mg/L SO 2 mass concentration, pH 3.2, 250 g/L sucrose mass concentration, and 300 g/L glucose mass concentration. Both strains HJ-2 and HJ-6 demonstrated strong ester production capability and low hydrogen sulfide production. Overall, strains HJ-2 and HJ-6 show potential for application in the industrial production of P. fortuneana wine. Pyracantha fortuneana Yeast Isolation and Identification Brewing characteristics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Pyracantha fortuneana ( P. fortuneana ), commonly known as firethorn , torch fruit , or rescue grain , is an evergreen wild shrub belonging to the genus Pyracantha Roem in the subfamily Maloideae of the Rosaceae family (Li et al. 2022 ). There are a total of 10 species of the Pyracantha genus worldwide, including Pyracantha fortuneana Li., Pyracantha atalantioides Stapf., Pyracantha crenulata Roem., Pyracantha angustifolia Schneid., Pyracantha densiflora Yü., Pyracantha inermis Vidal., Pyracantha koidzumii Rehd., Pyracantha crenulata var. kansuensis, Pyracantha coccinea , and Pyracantha coccinea var. lalandei, which are mainly distributed in East Asia and Southern Europe (Yao et al. 2020 ). As a dual-purpose wild fruit resource, P. fortuneana fruit is rich in nutritional components (Fig. 1 ). Studies have shown that P. fortuneana fruits contain amino acids, unsaturated fatty acids, and various mineral elements, which have effects such as delaying aging, maintaining blood pressure, and enhancing immunity. Regular consumption is beneficial to human health (Wang et al. 2020; Xu et al. 2013; Liu et al. 2023 ). With the improvement in living standards, a healthy lifestyle has guided the transformation of alcoholic beverage production from grain-based spirits to fruit wines (Liu et al. 2022 ). Due to the inherent lack of volatile phenolic compounds in P. fortuneana fruit, the flavor of the resulting P. fortuneana fruit wine is often insufficient. Therefore, the parameter settings and control management during the fermentation process of P. fortuneana fruit wine present significant challenges (Mo et al., 2019). Currently, research on P. fortuneana is mostly focused on areas such as ecology (Wu et al. 2023), bonsai (Chen et al. 2021), fresh fruit consumption (Hong et al. 2019), chemical engineering (Yao et al. 2020 ), and medicinal uses (Suárez-Lepe et al. 2012), with limited studies on the isolation, identification, and brewing characteristics of its yeast strains. Yeast plays a crucial role in the fermentation process of fruit wine, as its metabolic products influence the color, aroma, and taste of the wine, thereby determining the quality of the fruit wine (Hu et al. 2023 ). However, during the fruit wine brewing process, various factors such as pH, alcohol concentration, and temperature not only affect the growth and reproduction of yeast but may also cause fermentation to pause or terminate (Liu et al. 2020 ). Yeasts can be broadly categorized into two types: Saccharomyces cerevisiae and non- Saccharomyces . Non- Saccharomyces have poor alcohol metabolism capabilities but strong flavor compound production abilities, while wine yeasts, although limited in their capacity to produce various flavor compounds, exhibit strong fermentation performance and high alcohol production capabilities (Huang et al. 2022 ). In recent years, advancements in biotechnology have provided a solid foundation for the selection and breeding of superior yeast strains (Liao et al. 2020 ). The quality of yeast significantly impacts both the yield and quality of wine, as the characteristics of yeast directly affect the overall quality of fruit wine (Gong et al. 2023 ). Yeast selection is fundamental to the breeding of superior yeast strains; thus, isolating high-quality brewing yeast from P. fortuneana is a key step in producing high-quality, distinctive P. fortuneana fruit wine. In summary, this study used naturally fermented P. fortuneana fruit liquid as the isolation source, employing enrichment culture, purification, molecular biological identification, and tolerance tests to select local specialized yeast strains suitable for brewing P. fortuneana fruit wine, with the aim of laying a foundation for the improvement of the overall quality of P. fortuneana fruit wine. Materials and reagents P. fortuneana fruit: Collected from Liaokuo Mountain, Qilin District, Qujing City, Yunnan Province, September 2023. Commercial yeast (SY): Purchased from Angel Yeast Co., Ltd.; Fungicides, chloramphenicol, ampicillin, kanamycin, and agar powder (biochemical reagents): Beijing Solarbio Science & Technology Co., Ltd.; Glucose, sucrose, citric acid (food grade), sodium metabisulfite: ACS grade from Biological Engineering (Shanghai) Co., Ltd. Malt extract solid medium, yeast extract peptone dextrose agar medium (YPD), YPD liquid medium, and WL nutrient agar: Qingdao Hi-tech Industrial Park Haibo Biotechnology Co., Ltd. All media were sterilized at 121°C under high pressure steam for 15 minutes before use. BIGGY medium (bismuth sulfite glucose glycine yeast): Heated and boiled for no more than 1 minute, cooled to 45–50°C, poured into petri dishes without high-pressure sterilization. Experimental methods Preparation of P. fortuneana fruit fermentation liquid Remove diseased and spoiled P. fortuneana fruits, crush them, and place them in sterile Erlenmeyer flasks. Allow natural fermentation at 28°C until a wine-like aroma is observed, stirring the mixture twice daily (morning and evening). Isolation, purification, and morphological identification of yeast Take 1 ml of the fermentation liquid and perform a dilution plate method. Plate the diluted solution onto malt extract solid medium and YPD solid medium containing antibiotics, and incubate at 28°C for 24 to 48 hours. Observe colony growth and select single colonies with typical yeast characteristics for streak purification on YPD plates. After activation for 24 hours in YPD liquid medium, inoculate the isolated yeast strains into WL agar medium and incubate at 28°C for 5 days. Observe and record the colony color and morphology, and prepare water mount slides for microscopic observation. Analyze and preliminarily classify the yeast based on asexual reproduction and cell shape. Fermentation performance testing of yeast strains Place inverted Durham tubes into YPD liquid medium, sterilize at 121°C under high pressure for 15 minutes, then inoculate with the preliminary screened yeast strains. Mix thoroughly to remove air bubbles, and incubate in a 28°C constant temperature incubator. Observe and record gas production and sediment formation in the Durham tubes over 24 to 72 hours, and measure the gas volume. Sulfur hydrogen production characteristics of yeast strains Under sterile conditions, inoculate activated yeast strains onto the surface of BIGGY medium. Seal and incubate upside down at 28°C for 3 days. Observe changes in colony color. The color progression from light to dark is: white, light brown, dark brown, and black, indicating H 2 S production capability as none, low, moderate, and high, respectively (Zhou et al. 2020 ). Determination of ester production ability of yeast strains Under sterile conditions, inoculate the activated yeast strains onto the surface of YPD medium for ester production screening. Seal the plates and incubate upside down in a constant temperature incubator at 28°C for 3 days. Observe the size of the transparent zones around the colonies; a larger transparent zone indicates a stronger ability to produce esters, while a smaller transparent zone indicates a weaker ability. Molecular biological identification of yeast strains DNA extraction from strains: Inoculate the strain preserved in glycerol tubes into 5 mL of YPD liquid medium and culture on a shaking incubator at 180 r/min and 28°C for 14 hours, during which the strain is in the logarithmic growth phase. Collect the cells by centrifuging the culture in a 1.5 mL centrifuge tube (discard the supernatant). Use a fungal DNA extraction kit to extract the genomic DNA of the yeast as a template. PCR amplification: Based on references (Wu et al. 2015 ; Monnin et al. 2014), perform PCR amplification of the target strain using internal transcribed spacer (ITS) primers ITSI (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTC - CGCTTATTGATATGC-3'). The PCR amplification system consists of 45 µL of 1× PCR MIX, 2 µL of forward primer ITS1, 2 µL of reverse primer ITS4, and 1 µL of DNA template. PCR reaction conditions are as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 1 minute, annealing at 50°C for 1 minute, and extension at 72°C for 2 minutes, followed by 30 cycles and a final extension at 72°C for 10 minutes, then cooling down to 4°C to stop the reaction. After the reaction, take 5 µL of the PCR product and perform electrophoresis on a 1% agarose gel at 110 V. Visualize and photograph under a 260 nm UV lamp, and then send the sample to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing. Phylogenetic tree construction: Compare the sequencing results with regional sequences in the GenBank database using BLAST, construct a phylogenetic tree, and analyze the taxonomic status of the strain. Tolerance determination of yeast strains Under sterile conditions, inoculate the activated strains at a 2% inoculum level into YPD liquid medium with varying pH values of 2.8, 3.2, 3.6, and 4.0, ethanol concentrations of 3%, 6%, 9%, 12%, and 15%, temperatures of 4°C, 15°C, 25°C, 30°C, and 40°C, and sucrose and glucose concentrations of 200, 250, 300, 350, and 400 g/L, as well as SO 2 concentrations of 60, 120, 180, 240, and 300 mg/L. Perform three parallel repetitions and incubate at 28°C on a shaking incubator at 180 rpm for 24 hours. After incubation, measure the optical density (OD) of the strains at a wavelength of 600 nm using a UV spectrophotometer. Data processing and analysis The results are expressed as "mean ± standard deviation."Physicochemical parameters were analyzed using EXCEL 2021 software, and the phylogenetic tree was constructed using MEGA X software. Results and discussion Isolation and identification of yeast strains Yeast strains were isolated and purified from the naturally fermented wild pyracantha fruit fermentation liquid. After culturing these strains on WL nutrient agar, they were initially classified into 12 groups based on their surface morphology and colony color, numbered HJ-1 to HJ-12. Figure 2 displays the colony characteristics and microscopic features of the yeast strains grown on WL nutrient agar. It is evident that there are differences among these 12 yeast strains in terms of colony morphology, cytological characteristics, and reproduction methods. In terms of colony morphology, they exhibit irregular round shapes with colors ranging from white to green. Specifically, strain HJ-1 has a wavy edge; strains HJ-3, HJ-9, and HJ-12 have petal-like shapes; strains HJ-2, HJ-4, HJ-5, and HJ-8 display concentric rings; while strains HJ-6, HJ-7, HJ-10, and HJ-11 have fringed edges. In terms of cytological characteristics, the cells are round or oval, primarily reproducing by budding. Yeast fermentation performance Yeast fermentation primarily involves the conversion of sugars into alcohol and carbon dioxide. The rate of CO 2 production indicates the fermentation speed of the yeast (Wang et al. 2023 ). Within 24 hours, yeast strains that fill the Durham tubes with gas exhibit strong fermentation capabilities. Yeast strains with slower fermentation rates do not meet the requirements for industrial production, and prolonged fermentation of fruit wine increases the risk of microbial contamination. As shown in Table 1 , after 24 hours of fermentation, strains HJ-2 and HJ-3 produce gas and fill the Durham tubes, indicating the fastest gas production rate and the strongest fermentation ability. After 48 hours of fermentation, strains HJ-1, HJ-5, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 also fill the Durham tubes with gas, demonstrating relatively fast gas production and strong fermentation ability. After 72 hours of fermentation, strains HJ-4 and HJ-6 achieve gas volumes reaching 2/3 of the Durham tube, indicating slower gas production rates and weaker fermentation abilities. Table 1 Gas production in the Durcham tubes. Strain Number 24 h 48 h 72 h Gas Production Precipitation Status Gas Production Precipitation Status Gas Production Precipitation Status HJ−1 + White, Compact Precipitate ++ White, Compact Precipitate +++ White, Compact Precipitate HJ−2 +++ White, Compact Precipitate +++ White, Compact Precipitate +++ White, Compact Precipitate HJ−3 +++ White, Compact Precipitate +++ White, Compact Precipitate +++ White, Compact Precipitate HJ−4 ++ White, Compact Precipitate ++ White, Compact Precipitate ++ White, Compact Precipitate HJ−5 ++ White, Compact Precipitate +++ White, Compact Precipitate +++ White, Compact Precipitate HJ−6 ++ White, Compact Precipitate ++ White, Compact Precipitate ++ White, Compact Precipitate HJ-7 ++ White, Loose Precipitate +++ White, Loose Precipitate +++ White, Loose Precipitate HJ-8 + White, Loose Precipitate +++ White, Loose Precipitate +++ White, Loose Precipitate HJ-9 + White, Loose Precipitate +++ White, Loose Precipitate +++ White, Loose Precipitate HJ-10 + White, Loose Precipitate +++ White, Loose Precipitate +++ White, Loose Precipitate HJ-11 + White, Loose Precipitate +++ White, Loose Precipitate +++ White, Loose Precipitate HJ-12 ++ White, Loose Precipitate +++ White, Loose Precipitate +++ White, Loose Precipitate Note: "+, ++, +++" indicate that gas production reaches 1/3, 2/3, and all of the Duchenne tubule volume, respectively. H 2 S production capability Wine yeast produces trace amounts of H 2 S through sulfur metabolism, which has the odor of rotten eggs, garlic, or onions, negatively affecting the flavor profile of fruit wine. H 2 S can combine with bismuth to form a black bismuth sulfide precipitate, and the H 2 S production capability of the strains can be determined based on the color of the colonies on the selective medium BIGGY using bismuth as an indicator. Based on the levels of H 2 S production, the strains can be categorized into six levels: white, light brown, brown, light brown, brown, and black (Ying et al. 2024 ). As shown in the results of Fig. 3 , strains HJ-2, HJ-3, and HJ-5 display white and light brown colors, classifying them as low H 2 S producers; strains HJ-6 and HJ-4 exhibit deep brown colors, categorizing them as mediumH 2 S producers; while strains HJ-1, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 show dark brown colors, indicating them as high H 2 S producers. Zheng et al. ( 2020 ) isolated four indigenous wine yeast strains from grape-growing regions to assess their H 2 S production capabilities and found that strains WJ1, Q12, and S21 were medium H 2 S producers, while strain S12 was a high H2S producer. Huang et al. ( 2024 ) isolated six non-wine yeast strains from naturally fermented goat milk fruit, where strains H1, H7, and H10 were white, strain H5 had light brown edges, and strains H9 and H12 were deep brown. This indicates that strains H1, H7, and H10 do not produce H 2 S, strain H5 produces low levels of H 2 S, and strains H9 and H12 produce high levels of H 2 S. Compared to the above studies, this demonstrates certain differences in the H 2 S production capabilities of yeast, further emphasizing the importance of yeast selection. Ester production capability Microbial secreted enzymes can catalyze the reaction between acids and alcohols to produce esters, which are further hydrolyzed into acids and alcohols, thereby enhancing the flavor of fruit wine (Yuan et al. 2023 ). By inoculating yeast strains into a medium containing butyric glycerol ester and utilizing the esterases produced by the yeast to hydrolyze butyric glycerol ester, a transparent zone is formed that can be used to evaluate the ester production capability of the yeast strains (Ying et al. 2024 ). As shown in Fig. 4 , strains HJ-5, HJ-6, HJ-11, HJ-3, HJ-2, and HJ-8 exhibit significant transparent zones, indicating strong ester production capabilities; strains HJ-4, HJ-10, HJ-7, and HJ-12 have smaller transparent zones, indicating weaker ester production capabilities; while strains HJ-9 and HJ-1 show no obvious transparent zones, suggesting that these two yeast strains lack ester production capability. Long et al. ( 2024 ) assessed the ester production capabilities of five wine yeast strains from the fermentation liquor of Dian olive and found that the five strains exhibited varying ester production abilities, with strain LJM-10 demonstrating a strong ester production capacity. This indicates that there are certain differences in the ester production capabilities among different yeast strains. Molecular biological identification The 26S rDNA D1/D2 region within ribosomal DNA is commonly used for the molecular biological classification of yeast (Wang et al. 2024 ). PCR amplification was performed on the 12 selected yeast strains, and as shown in Fig. 5 , the size of the 26S rDNA D1/D2 region in all 12 strains is approximately 600 bp, which matches the expected size. The sequencing results were compared with the NCBI database using BLAST (see Table 2 ), and a phylogenetic tree was constructed (see Fig. 6 ). Strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 showed over 98% similarity to known strains of Pichia kudriavzevii AC1 (OP678979.1), Pichia kudriavzevii B-NC-13-OZ23 (KJ794697.1), Pichia kudriavzevii 11 (KR259307.1), Pichia kudriavzevii YZ4 (EU394711.1), Pichia kudriavzevii PEX-11 (MW990004.1), Pichia kudriavzevii 15 (KR259308.1), Pichia kudriavzevii L1 (EF126365.1), Pichia kudriavzevii feni48 (KM234455.1) and clustered with Pichia kudriavzevii CDA174Y2 (OQ568324.1), indicating that strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 belong to Pichia kudriavzevii . Strains HJ-2, HJ-3, HJ-4, and HJ-5 showed over 99% similarity to known strains of Saccharomyces cerevisiae YC-D8 (OP644089.1), Saccharomyces cerevisiae QTX-D20 (OP644141.1), Saccharomyces cerevisiae QTX-D14 (OP644135.1), Saccharomyces cerevisiae YQY-F11 (OP644252.1)) and clustered with Saccharomyces cerevisiae UTAD97 (OQ305072.1), indicating that strains HJ-2, HJ-3, HJ-4, and HJ-5 belong to Saccharomyces cerevisiae . It is widely recognized that strains with the same genotype tend to exhibit similar brewing characteristics (Bi et al. 2023). Therefore, one strain from Pichia kudriavzevii and one from Saccharomyces cerevisiae with good fermentation performance, low H 2 S production, and high ester production (HJ-2, HJ-6) were selected for tolerance testing. Table 2 Sequencing results of 26S rDNA D1/D2 sequence of five strain of yeast. Strain Code Series Length Homologous Strain Homologous strain number degree of similarity(%) Genebanknumber HJ−1 575 bp Pichia kudriavzevii AC1 99% OP678979.1 HJ−2 585 bp Saccharomyces cerevisiae YC-D8 99% OP644089.1 HJ−3 589 bp Saccharomyces cerevisiae QTX-D20 100% OP644141.1 HJ−4 587 bp Saccharomyces cerevisiae QTX-D14 100% OP644135.1 HJ−5 585 bp Saccharomyces cerevisiae YQY-F11 100% OP644252.1 HJ−6 577 bp Pichia kudriavzevii B-NC−13-OZ23 98% KJ794697.1 HJ−7 582 bp Pichia kudriavzevii 11 100% KR259307.1 HJ−8 581 bp Pichia kudriavzevii YZ4 99% EU394711.1 HJ−9 565 bp Pichia kudriavzevii PEX−11 99% MW990004.1 HJ−10 582 bp Pichia kudriavzevii 15 99% KR259308.1 HJ−11 570 bp Pichia kudriavzevii L1 99% EF126365.1 HJ−12 580 bp Pichia kudriavzevii feni48 99% KM234455.1 Tolerance of yeast strains The pH of fruit wine typically ranges from 3.0 to 4.0, which necessitates the use of yeast with strong acid tolerance during the winemaking process; both excessively high and low pH levels can affect yeast activity (Ying et al. 2024 ). Therefore, in this study, commercial yeast SY was used as a control strain to assess the pH tolerance of the selected strains HJ-2 and HJ-6. The results are shown in Fig. 7 (A), where it can be observed that as the environmental acidity increases, the growth condition of the strains gradually weakens. At a pH of 2.8, commercial yeast SY was able to grow normally, while the OD 600 nm values of strains HJ-2 and HJ-6 were below 0.5, approaching zero, indicating that the increased acidity significantly inhibited their growth. Conversely, as the acidity level decreased from pH 3.2 to pH 4.0, the growth rates of commercial yeast SY and strains HJ-2 and HJ-6 showed an upward trend, indicating their effective adaptability to these conditions. This finding is consistent with the results of Long et al. ( 2024 ), which studied the pH tolerance of five yeast strains. Therefore, it can be concluded that strains HJ-2 and HJ-6 exhibit good tolerance to low pH environments. Ethanol is the primary metabolic product of yeast, and the accumulation of excessive ethanol in fruit wine fermentation can exert inhibitory and toxic effects on the yeast involved in the fermentation process, potentially leading to yeast cell death. The ethanol tolerance of yeast is closely related to its fermentation performance (Gan et al., 2022 ). The ethanol tolerance of strains HJ-2, HJ-6, and commercial yeast SY was assessed, and the results are shown in Fig. 7 (B). Within the ethanol concentration range of 3–15%, the OD values of the yeast generally exhibited a downward trend as ethanol concentration increased. At an ethanol concentration of 3%, strains SY, HJ-2, and HJ-6 were able to grow normally. However, when the ethanol concentrations reached 6% and 9%, the growth of strain HJ-6 was inhibited, while strains SY and HJ-2 continued to grow normally. When the ethanol concentration increased to 12% and 15%, only strain HJ-2 grew normally, while the growth of strains SY and HJ-6 was inhibited. These results indicate that strain HJ-2 can tolerate ethanol concentrations of up to 15%, whereas the tolerance limits for strains SY and HJ-6 are 9% and 3%, respectively. Qin et al. ( 2022 ) found that the ethanol tolerance of the strain Hyphopichia burtonii Y9 was 9%, categorizing it as a strain with moderate ethanol tolerance. Additionally, Jiang et al. ( 2024 ) reported that five aromatic yeast strains selected from high-temperature Daqu exhibited ethanol tolerances of 7%. Comparing these findings, it can be concluded that the selected strain HJ-2 demonstrates high ethanol tolerance, making it a suitable strain for the production of hawthorn fruit wine. Sugar is the primary substrate for ethanol production through fermentation and serves as the energy source for yeast. However, high sugar concentrations can inhibit yeast growth and reproduction, as high osmotic pressure may lead to water loss in yeast cells (Zhou et al. 2024 ). The glucose and sucrose tolerance of strains SY, HJ-2, and HJ-6 were tested, with results shown in Fig. 7 (C) and Fig. 7 (D). The commercial yeast SY exhibited minimal growth impact under various glucose and sucrose concentrations. Strain HJ-6 demonstrated tolerance when glucose concentration reached 300 g/L, while strain HJ-2 experienced varying degrees of inhibition as glucose concentration increased. When sucrose was used as the carbon source, strain HJ-2 maintained an OD 600 nm value greater than 1.0 at different sucrose concentrations, indicating strong tolerance to sucrose levels up to 400 g/L. These results highlight significant differences in sugar tolerance between strains HJ-2 and HJ-6 under different carbon sources. Zhao et al. ( 2023 ) isolated the strain F3 from naturally fermented mulberry juice, which exhibited the best tolerance, being able to withstand a maximum sugar concentration of 400 g/L. Mou et al. ( 2023 ) identified excellent strains B-6 and C-3 from Qingxiang Daqu that could tolerate glucose concentrations of 400 g/L. Compared to the aforementioned studies, the strains HJ-2 and HJ-6 selected in this research demonstrated good sugar tolerance. In wine production, the addition of SO 2 in appropriate amounts can inhibit harmful microorganisms and provide antioxidant and color-preserving effects. However, higher concentrations of SO 2 can adversely affect yeast strain growth, thereby prolonging fermentation time, and high osmotic pressure can also impact the metabolic products of yeast during alcoholic fermentation (Hu et al. 2023 ). As shown in Fig. 7 (E), strains HJ-2, HJ-6, and SY exhibited normal growth under different SO 2 concentrations, with no significant changes, indicating that these strains can tolerate SO 2 concentrations up to 360 mg/L. Gong et al. ( 2023 ) identified four yeast strains, YC-E8, QTX-D17, QTX-D7, and YQY-E18, from 168 local brewing yeast strains in Ningxia, which could tolerate 250 mg/L SO 2 . Zhang et al. ( 2022 ) isolated three excellent yeast strains, GL14, GP21, and GP24, from prickly pear, all of which could tolerate 300 mg/L SO 2 . These findings indicate that strains HJ-2 and HJ-6 exhibit good SO 2 tolerance, meeting the standards for high-quality yeast strains. Temperature has a significant impact on yeast reproduction, with the optimal growth temperature for yeast being between 25°C and 30°C. Temperatures exceeding 35°C can lead to cell death; however, certain yeast strains can survive at temperatures above 40°C (Lai et al. 2022 ). In this study, temperature tolerance tests were conducted on the selected strains, as shown in Fig. 7 (F), which displays the temperature tolerance of the three yeast strains. Under conditions of 4°C, 15°C, 25°C, 30°C, and 40°C, the OD values of the yeast strains initially increased and then decreased. At 4°C, the growth of commercial yeast SY, as well as strains HJ-2 and HJ-6, was inhibited. At 15°C and 25°C, strains SY and HJ-2 grew without inhibition, while strain HJ-6 was inhibited. At 30°C, all three yeast strains exhibited normal growth. At 40°C, strains SY and HJ-2 demonstrated better growth compared to strain HJ-6. These results indicate that the optimal temperature for strains SY, HJ-2, and HJ-6 is 30°C, with the ability to tolerate high temperatures of up to 40°C, which is consistent with the heat resistance findings for industrial yeast strains at 40°C. Conclusion In this study, 12 yeast strains were isolated from the fermentation liquid of wild thorny fruits. Based on 26S rDNA D1/D2 region gene sequence analysis, strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 were identified as Pichia kudriavzevii , while strains HJ-2, HJ-3, HJ-4, and HJ-5 were identified as Saccharomyces cerevisiae . Two strains (HJ-2 and HJ-6) were selected for tolerance testing due to their good fermentation performance, high ester production, and low H 2 S production. Strain HJ-2 was able to grow normally under the following conditions: 40°C, 15% (v/v) ethanol, 360 mg/L SO 2 , pH 3.2, 400 g/L sucrose, and 250 g/L glucose. Strain HJ-6 grew normally under the conditions of 40°C, 3% (v/v) ethanol, 360 mg/L SO 2 , pH 3.2, 250 g/L sucrose, and 300 g/L glucose. Both strains HJ-2 and HJ-6 exhibited strong ester production capabilities and low H 2 S production. Gan et al. ( 2022 ) screened a strain of brewing yeast that could withstand pH 1.6, 18.2% ethanol concentration, 45°C temperature, and 250 mg/L SO 2 . Tan et al. ( 2020 ) isolated two brewing yeast strains from ten yeast strains that could grow and reproduce under conditions of 16% ethanol, 500 g/L glucose, 250 mg/L SO 2 , high temperature of 45°C, and low temperature of 5°C. Lei et al. ( 2024 ) isolated five non-brewing yeast strains with strong ester production capabilities from naturally fermented juice, among which strain Wa3 showed the best tolerance to high concentrations of alcohol, sugar, and SO 2 . Comparing with the above studies, strains HJ-2 and HJ-6 demonstrate excellent characteristics for fruit wine brewing. In summary, strains HJ-2 and HJ-6 are expected to provide yeast resources for fruit wine production and possess significant application potential in the thorny fruit wine industry. Declarations Author contributions The experiments were designed and conducted by L. Z., Data analysis was performed by J. Y. Y., Q. F. X., X. B., X. G., L. F. Z., W. L. L., H. H. W., and L. Z. The project was conceived and supervised by L. Z. The paper was written by L. Z. and J. C. with contributions from all authors, in accordance with the approval of the manuscript. Conflicts of interest The authors declare that there are no conficts of interest. Funding information This work was supported by Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (Grant NO. 202301BA070001-081), Yunnan Fundamental Research Projects (Grant NO. 202301AU070012), Special Basic Cooperative Research Innovation Programs of Qujing Science and Technology Bureau & Qujing Normal University (Grant NO. KJLH2023ZD06, KJLH2022YB07), National College Students Innovation and Entrepreneurship Training Program, China (Grant NO. G202310684015, G202310684002). Ethical approval No experiments involving human participants or animals were conducted by any of the authors in this study. References Bi JY, Lu LJ (2023) Screening of native Saccharomyces cerevisiae and its application in navel orange wine brewing. 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Wang Y, Shao QJ, Yang XJ, Su K, Li ZR, Yang YY, Yuan XY, Chen RX (2024) Diversity in Pyracantha fortuneana fruits maturity stages enables discrepancy in the phenolic compounds, antioxidant activity, and tyrosinase inhibitory activity. J Food Sci 1-15. Wu JR, Wang XR, Tang XY, Wang QQ, Wu RN, Yue XQ (2015) Identification of lactic acid bacteria and yeast from naturally fermented soybean paste from Liaoning province. Food Sci 36 : 78-83. Wu YC, Yan YT (2023) Research on landscaping application and cultivation management techniques of Pyracantha fortuneana. Hortic Seed 43 : 49-50. Xu F, Zha CC (2013) Study on the in vivo immune activity of red pigment extract from Pyracantha fruit. Guide China Med 11 : 97-98. Yao YL, Shu C, Feng G, Wang Q, Yan YY, Yi Y, Wang HX, Zhang XF, Wang LM (2020) Polysaccharides from Pyracantha fortuneana and its biological activity. Int J Biol Macromol 150 : 1162-1174. Ying BB, Cai J, Gao X, Zhang LF, Xu QF, Xu QH, Liu WL, Huang XM, Wang YC, Zhu L (2024) Isolation, identification, and tolerance analysis of yeast during the natural fermentation process of Sidamo coffee beans. Arch Microbiol 206 : 1-12. Yuan HS, Liu GL, Bai WD, Xiao GS, Liang JL (2023) Research progress on the application of ester-producing yeast in fermented food. China Brew 42 : 15-20 Zhang XQ, Pan XS, Li D, Jiang SX, Zhang F, An YL, Zhao JJ, Song Y, Yu SR, Zhang Y, Yan RY (2022) Screening and fermenting property of alcohol-producing yeasts in Rosa roxbunghii from Guizhou. China Brew 41 : 97-102. Zhao ST, Ding B, Xiong L, Lin Y, Wu L, Tong LH, Zhang WX (2023) Screening of fragrant yeast and analysis of fermentation characteristics of mulberry Wine. Food Sci. Technol 48 : 9-15. Zheng HW, Lei L, Li ZY, Zhao. XP, Zhang MZ, Li T, Huang HY, Li XJ, Wang CY (2020) Oenological screening for high-quality Chinese Saccharomyces cerevisiae strains. Food Ferment Ind 46 : 118-130. Zhou D, Lu L, Wang L (2024) Screening and fermentation characteristics of non- Saccharomyces yeasts from eastern foot of Helan Mountain Food Ferment Ind 1-11. Zhou YZ, Bian MH, Liu WY, Jiang JJ, Xu WJ (2020) Isolation an identification of an amylase producing yeast and its characteristics in high-temperature Daqu. Food Ferment Ind 46 : 79-84. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Oct, 2024 Read the published version in Archives of Microbiology → Version 1 posted Editorial decision: Revision requested 16 Aug, 2024 Reviews received at journal 14 Aug, 2024 Reviews received at journal 11 Aug, 2024 Reviewers agreed at journal 10 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers agreed at journal 07 Aug, 2024 Reviewers agreed at journal 06 Aug, 2024 Reviewers agreed at journal 05 Aug, 2024 Reviewers agreed at journal 05 Aug, 2024 Reviewers invited by journal 05 Aug, 2024 Editor assigned by journal 02 Aug, 2024 Submission checks completed at journal 01 Aug, 2024 First submitted to journal 01 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4842597","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":340980915,"identity":"3e510e78-c1f2-4c42-b840-757b652daff2","order_by":0,"name":"Ling Zhu","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Zhu","suffix":""},{"id":340980916,"identity":"3eaca065-e53e-46ea-aa6b-f46444a997ba","order_by":1,"name":"Jiang-Yan Yu","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Jiang-Yan","middleName":"","lastName":"Yu","suffix":""},{"id":340980918,"identity":"c9ea6715-ddab-43c4-90a4-b12273b5e2b2","order_by":2,"name":"Qing-Fang Xu","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Qing-Fang","middleName":"","lastName":"Xu","suffix":""},{"id":340980919,"identity":"ab33bf07-da38-4d2c-8d82-1c0a37b9d645","order_by":3,"name":"Xu Bai","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Bai","suffix":""},{"id":340980921,"identity":"e49e93b6-9a47-44ca-8a78-e204ed809238","order_by":4,"name":"Xiu Gao","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Xiu","middleName":"","lastName":"Gao","suffix":""},{"id":340980924,"identity":"2c6140a2-e5d9-42f8-98d6-cbc8affeba7c","order_by":5,"name":"Li-Fang Zhang","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Li-Fang","middleName":"","lastName":"Zhang","suffix":""},{"id":340980926,"identity":"221409c2-9531-4445-a343-3f5d0f68d6a1","order_by":6,"name":"Wei-Liang Liu","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Wei-Liang","middleName":"","lastName":"Liu","suffix":""},{"id":340980928,"identity":"1f6be84e-8b1e-41b5-a6cc-b8ac09268648","order_by":7,"name":"Hao-Han Wang","email":"","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":false,"prefix":"","firstName":"Hao-Han","middleName":"","lastName":"Wang","suffix":""},{"id":340980929,"identity":"d37781c9-98d2-4341-b4ef-36ba11fda731","order_by":8,"name":"Jian Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYFACxgaDBAYGOX725gNAngUDAw+RWowle44BqQQJYrRAQOKGGzkGxGnhbz/cUPCg5k5iw5kzHx/z/pCQ4+c5wPjhYw5uLRJnEoEOO/bMuLG9d7MxT4KEsWRvA7PkzG24tRgwgLSwHZZt5jm7TRqoJXHDeQY2Zl58WvgfArX8O8zYJpHzjEgtEkBbEtsOK/ZI5LBBtJxtwK9F4gbQlsS+w8YSPMeMDeekAf3Sc7AZr1/4+9OfGf74dljO/njzwwdvbGyAIZZ88MNHPFqAgM0ATYCxAa96IGB+QEjFKBgFo2AUjHAAAJaUUx+2EE9QAAAAAElFTkSuQmCC","orcid":"","institution":"Yunnan Engineering Research Center of Fruit Wine, Qujing Normal University, Qujing 655011, Yunnan, China","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2024-08-01 13:39:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4842597/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4842597/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00203-024-04164-4","type":"published","date":"2024-10-18T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63568400,"identity":"15e013a3-0416-498a-9eb4-79fa094f54c0","added_by":"auto","created_at":"2024-08-29 16:45:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":883403,"visible":true,"origin":"","legend":"\u003cp\u003eWild Firethorn Resources. Scale bar = 1 pixel.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/4151a29fe6d0498f4ca721d3.png"},{"id":63568034,"identity":"db7376e6-4068-4044-ac10-32bb5da02b40","added_by":"auto","created_at":"2024-08-29 16:37:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":250707,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological features of yeast. Scale bar = 100 pixel.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/0eedf25e7039e5f1ca83ed33.png"},{"id":63568039,"identity":"44878369-d879-4531-914e-c472b97be23e","added_by":"auto","created_at":"2024-08-29 16:37:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185001,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS production capacity of 12 yeast strains. Scale bar = 2 pixel.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/0c641c296649fe1b9caa4e56.png"},{"id":63568690,"identity":"8bb71355-7870-489c-8264-6f7b0dd5d447","added_by":"auto","created_at":"2024-08-29 16:53:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":124731,"visible":true,"origin":"","legend":"\u003cp\u003eEster production capacity of 12 yeast strains. Scale bar = 2 pixel.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/a81e754abc46dfa30cc5d62d.png"},{"id":63568032,"identity":"e043e995-0314-467d-be5f-947cec57d4d9","added_by":"auto","created_at":"2024-08-29 16:37:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135599,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoretic map of the sequence PCR products of isolate 26S rDNA D1/D2 of five strains of yeast. Scale bar = 1 pixel.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/5b2996032f1ed7b39fdd4941.png"},{"id":63568401,"identity":"2adee2d1-fea7-4888-9026-24287a6809bb","added_by":"auto","created_at":"2024-08-29 16:45:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":27661,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of yeast strains was conducted based on the 26S rDNA sequence.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/3480eedbbae60645994aa910.png"},{"id":63568038,"identity":"53527440-827f-41bc-873c-c7323cb29683","added_by":"auto","created_at":"2024-08-29 16:37:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":96564,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristics of yeast tolerance.\u003cstrong\u003e (A) \u003c/strong\u003epH tolerance,\u003cstrong\u003e (B) \u003c/strong\u003eEthanol tolerance,\u003cstrong\u003e (C) \u003c/strong\u003eGlucose tolerance,\u003cstrong\u003e (D) \u003c/strong\u003eSucrose tolerance, \u003cstrong\u003e(E) \u003c/strong\u003eSO\u003csub\u003e2\u003c/sub\u003e tolerance,\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e (F) \u003c/strong\u003eTemperature tolerance.\u003cstrong\u003e \u003c/strong\u003eDifferent lowercase letters above the standard deviation bar indicate a significant difference (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/60e1871b0a119013398c1c35.png"},{"id":67149779,"identity":"eb38920c-5a28-4347-8dd8-dbf015861778","added_by":"auto","created_at":"2024-10-21 16:14:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2726087,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4842597/v1/6d251c97-e757-4905-b99b-3bf6ebdea33d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePyracantha fortuneana\u003c/em\u003e (\u003cem\u003eP. fortuneana\u003c/em\u003e), commonly known as \u003cem\u003efirethorn\u003c/em\u003e, \u003cem\u003etorch fruit\u003c/em\u003e, or \u003cem\u003erescue grain\u003c/em\u003e, is an evergreen wild shrub belonging to the genus \u003cem\u003ePyracantha Roem\u003c/em\u003e in the subfamily \u003cem\u003eMaloideae\u003c/em\u003e of the \u003cem\u003eRosaceae\u003c/em\u003e family (Li et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). There are a total of 10 species of the \u003cem\u003ePyracantha\u003c/em\u003e genus worldwide, including \u003cem\u003ePyracantha fortuneana\u003c/em\u003e Li., \u003cem\u003ePyracantha atalantioides\u003c/em\u003e Stapf., \u003cem\u003ePyracantha crenulata\u003c/em\u003e Roem., \u003cem\u003ePyracantha angustifolia\u003c/em\u003e Schneid., \u003cem\u003ePyracantha densiflora\u003c/em\u003e Y\u0026uuml;., \u003cem\u003ePyracantha inermis\u003c/em\u003e Vidal., \u003cem\u003ePyracantha koidzumii\u003c/em\u003e Rehd., \u003cem\u003ePyracantha crenulata\u003c/em\u003e var. kansuensis, \u003cem\u003ePyracantha coccinea\u003c/em\u003e, and \u003cem\u003ePyracantha coccinea\u003c/em\u003e var. lalandei, which are mainly distributed in East Asia and Southern Europe (Yao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a dual-purpose wild fruit resource, \u003cem\u003eP. fortuneana\u003c/em\u003e fruit is rich in nutritional components (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Studies have shown that \u003cem\u003eP. fortuneana\u003c/em\u003e fruits contain amino acids, unsaturated fatty acids, and various mineral elements, which have effects such as delaying aging, maintaining blood pressure, and enhancing immunity. Regular consumption is beneficial to human health (Wang et al. 2020; Xu et al. 2013; Liu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith the improvement in living standards, a healthy lifestyle has guided the transformation of alcoholic beverage production from grain-based spirits to fruit wines (Liu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Due to the inherent lack of volatile phenolic compounds in \u003cem\u003eP. fortuneana\u003c/em\u003e fruit, the flavor of the resulting \u003cem\u003eP. fortuneana\u003c/em\u003e fruit wine is often insufficient. Therefore, the parameter settings and control management during the fermentation process of \u003cem\u003eP. fortuneana\u003c/em\u003e fruit wine present significant challenges (Mo et al., 2019). Currently, research on \u003cem\u003eP. fortuneana\u003c/em\u003e is mostly focused on areas such as ecology (Wu et al. 2023), bonsai (Chen et al. 2021), fresh fruit consumption (Hong et al. 2019), chemical engineering (Yao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and medicinal uses (Su\u0026aacute;rez-Lepe et al. 2012), with limited studies on the isolation, identification, and brewing characteristics of its yeast strains.\u003c/p\u003e \u003cp\u003eYeast plays a crucial role in the fermentation process of fruit wine, as its metabolic products influence the color, aroma, and taste of the wine, thereby determining the quality of the fruit wine (Hu et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, during the fruit wine brewing process, various factors such as pH, alcohol concentration, and temperature not only affect the growth and reproduction of yeast but may also cause fermentation to pause or terminate (Liu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Yeasts can be broadly categorized into two types: \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and non-\u003cem\u003eSaccharomyces\u003c/em\u003e. Non-\u003cem\u003eSaccharomyces\u003c/em\u003e have poor alcohol metabolism capabilities but strong flavor compound production abilities, while wine yeasts, although limited in their capacity to produce various flavor compounds, exhibit strong fermentation performance and high alcohol production capabilities (Huang et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In recent years, advancements in biotechnology have provided a solid foundation for the selection and breeding of superior yeast strains (Liao et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The quality of yeast significantly impacts both the yield and quality of wine, as the characteristics of yeast directly affect the overall quality of fruit wine (Gong et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Yeast selection is fundamental to the breeding of superior yeast strains; thus, isolating high-quality brewing yeast from \u003cem\u003eP. fortuneana\u003c/em\u003e is a key step in producing high-quality, distinctive \u003cem\u003eP. fortuneana\u003c/em\u003e fruit wine.\u003c/p\u003e \u003cp\u003eIn summary, this study used naturally fermented \u003cem\u003eP. fortuneana\u003c/em\u003e fruit liquid as the isolation source, employing enrichment culture, purification, molecular biological identification, and tolerance tests to select local specialized yeast strains suitable for brewing \u003cem\u003eP. fortuneana\u003c/em\u003e fruit wine, with the aim of laying a foundation for the improvement of the overall quality of \u003cem\u003eP. fortuneana\u003c/em\u003e fruit wine.\u003c/p\u003e"},{"header":"Materials and reagents","content":"\u003cp\u003e \u003cem\u003eP. fortuneana\u003c/em\u003e fruit: Collected from Liaokuo Mountain, Qilin District, Qujing City, Yunnan Province, September 2023.\u003c/p\u003e \u003cp\u003eCommercial yeast (SY): Purchased from Angel Yeast Co., Ltd.; Fungicides, chloramphenicol, ampicillin, kanamycin, and agar powder (biochemical reagents): Beijing Solarbio Science \u0026amp; Technology Co., Ltd.; Glucose, sucrose, citric acid (food grade), sodium metabisulfite: ACS grade from Biological Engineering (Shanghai) Co., Ltd.\u003c/p\u003e \u003cp\u003eMalt extract solid medium, yeast extract peptone dextrose agar medium (YPD), YPD liquid medium, and WL nutrient agar: Qingdao Hi-tech Industrial Park Haibo Biotechnology Co., Ltd. All media were sterilized at 121\u0026deg;C under high pressure steam for 15 minutes before use.\u003c/p\u003e \u003cp\u003eBIGGY medium (bismuth sulfite glucose glycine yeast): Heated and boiled for no more than 1 minute, cooled to 45\u0026ndash;50\u0026deg;C, poured into petri dishes without high-pressure sterilization.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental methods\u003c/h2\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eP. fortuneana\u003c/b\u003e \u003cb\u003efruit fermentation liquid\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRemove diseased and spoiled \u003cem\u003eP. fortuneana\u003c/em\u003e fruits, crush them, and place them in sterile Erlenmeyer flasks. Allow natural fermentation at 28\u0026deg;C until a wine-like aroma is observed, stirring the mixture twice daily (morning and evening).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolation, purification, and morphological identification of yeast\u003c/h2\u003e \u003cp\u003eTake 1 ml of the fermentation liquid and perform a dilution plate method. Plate the diluted solution onto malt extract solid medium and YPD solid medium containing antibiotics, and incubate at 28\u0026deg;C for 24 to 48 hours. Observe colony growth and select single colonies with typical yeast characteristics for streak purification on YPD plates. After activation for 24 hours in YPD liquid medium, inoculate the isolated yeast strains into WL agar medium and incubate at 28\u0026deg;C for 5 days. Observe and record the colony color and morphology, and prepare water mount slides for microscopic observation. Analyze and preliminarily classify the yeast based on asexual reproduction and cell shape.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFermentation performance testing of yeast strains\u003c/h2\u003e \u003cp\u003ePlace inverted Durham tubes into YPD liquid medium, sterilize at 121\u0026deg;C under high pressure for 15 minutes, then inoculate with the preliminary screened yeast strains. Mix thoroughly to remove air bubbles, and incubate in a 28\u0026deg;C constant temperature incubator. Observe and record gas production and sediment formation in the Durham tubes over 24 to 72 hours, and measure the gas volume.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSulfur hydrogen production characteristics of yeast strains\u003c/h2\u003e \u003cp\u003eUnder sterile conditions, inoculate activated yeast strains onto the surface of BIGGY medium. Seal and incubate upside down at 28\u0026deg;C for 3 days. Observe changes in colony color. The color progression from light to dark is: white, light brown, dark brown, and black, indicating H\u003csub\u003e2\u003c/sub\u003eS production capability as none, low, moderate, and high, respectively (Zhou et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of ester production ability of yeast strains\u003c/h2\u003e \u003cp\u003eUnder sterile conditions, inoculate the activated yeast strains onto the surface of YPD medium for ester production screening. Seal the plates and incubate upside down in a constant temperature incubator at 28\u0026deg;C for 3 days. Observe the size of the transparent zones around the colonies; a larger transparent zone indicates a stronger ability to produce esters, while a smaller transparent zone indicates a weaker ability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMolecular biological identification of yeast strains\u003c/h2\u003e \u003cp\u003eDNA extraction from strains: Inoculate the strain preserved in glycerol tubes into 5 mL of YPD liquid medium and culture on a shaking incubator at 180 r/min and 28\u0026deg;C for 14 hours, during which the strain is in the logarithmic growth phase. Collect the cells by centrifuging the culture in a 1.5 mL centrifuge tube (discard the supernatant). Use a fungal DNA extraction kit to extract the genomic DNA of the yeast as a template.\u003c/p\u003e \u003cp\u003ePCR amplification: Based on references (Wu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Monnin et al. 2014), perform PCR amplification of the target strain using internal transcribed spacer (ITS) primers ITSI (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTC - CGCTTATTGATATGC-3'). The PCR amplification system consists of 45 \u0026micro;L of 1\u0026times; PCR MIX, 2 \u0026micro;L of forward primer ITS1, 2 \u0026micro;L of reverse primer ITS4, and 1 \u0026micro;L of DNA template. PCR reaction conditions are as follows: pre-denaturation at 95\u0026deg;C for 5 minutes; denaturation at 95\u0026deg;C for 1 minute, annealing at 50\u0026deg;C for 1 minute, and extension at 72\u0026deg;C for 2 minutes, followed by 30 cycles and a final extension at 72\u0026deg;C for 10 minutes, then cooling down to 4\u0026deg;C to stop the reaction. After the reaction, take 5 \u0026micro;L of the PCR product and perform electrophoresis on a 1% agarose gel at 110 V. Visualize and photograph under a 260 nm UV lamp, and then send the sample to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing.\u003c/p\u003e \u003cp\u003ePhylogenetic tree construction: Compare the sequencing results with regional sequences in the GenBank database using BLAST, construct a phylogenetic tree, and analyze the taxonomic status of the strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTolerance determination of yeast strains\u003c/h2\u003e \u003cp\u003eUnder sterile conditions, inoculate the activated strains at a 2% inoculum level into YPD liquid medium with varying pH values of 2.8, 3.2, 3.6, and 4.0, ethanol concentrations of 3%, 6%, 9%, 12%, and 15%, temperatures of 4\u0026deg;C, 15\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, and 40\u0026deg;C, and sucrose and glucose concentrations of 200, 250, 300, 350, and 400 g/L, as well as SO\u003csub\u003e2\u003c/sub\u003e concentrations of 60, 120, 180, 240, and 300 mg/L. Perform three parallel repetitions and incubate at 28\u0026deg;C on a shaking incubator at 180 rpm for 24 hours. After incubation, measure the optical density (OD) of the strains at a wavelength of 600 nm using a UV spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData processing and analysis\u003c/h2\u003e \u003cp\u003eThe results are expressed as \"mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\"Physicochemical parameters were analyzed using EXCEL 2021 software, and the phylogenetic tree was constructed using MEGA X software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and identification of yeast strains\u003c/h2\u003e \u003cp\u003eYeast strains were isolated and purified from the naturally fermented wild pyracantha fruit fermentation liquid. After culturing these strains on WL nutrient agar, they were initially classified into 12 groups based on their surface morphology and colony color, numbered HJ-1 to HJ-12. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e displays the colony characteristics and microscopic features of the yeast strains grown on WL nutrient agar. It is evident that there are differences among these 12 yeast strains in terms of colony morphology, cytological characteristics, and reproduction methods. In terms of colony morphology, they exhibit irregular round shapes with colors ranging from white to green. Specifically, strain HJ-1 has a wavy edge; strains HJ-3, HJ-9, and HJ-12 have petal-like shapes; strains HJ-2, HJ-4, HJ-5, and HJ-8 display concentric rings; while strains HJ-6, HJ-7, HJ-10, and HJ-11 have fringed edges. In terms of cytological characteristics, the cells are round or oval, primarily reproducing by budding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eYeast fermentation performance\u003c/h2\u003e \u003cp\u003eYeast fermentation primarily involves the conversion of sugars into alcohol and carbon dioxide. The rate of CO\u003csub\u003e2\u003c/sub\u003e production indicates the fermentation speed of the yeast (Wang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Within 24 hours, yeast strains that fill the Durham tubes with gas exhibit strong fermentation capabilities. Yeast strains with slower fermentation rates do not meet the requirements for industrial production, and prolonged fermentation of fruit wine increases the risk of microbial contamination. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, after 24 hours of fermentation, strains HJ-2 and HJ-3 produce gas and fill the Durham tubes, indicating the fastest gas production rate and the strongest fermentation ability. After 48 hours of fermentation, strains HJ-1, HJ-5, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 also fill the Durham tubes with gas, demonstrating relatively fast gas production and strong fermentation ability. After 72 hours of fermentation, strains HJ-4 and HJ-6 achieve gas volumes reaching 2/3 of the Durham tube, indicating slower gas production rates and weaker fermentation abilities.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGas production in the Durcham tubes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e48 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e72 h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGas Production\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrecipitation Status\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGas Production\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePrecipitation Status\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGas Production\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePrecipitation Status\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Compact Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ-12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWhite, Loose Precipitate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: \"+, ++, +++\" indicate that gas production reaches 1/3, 2/3, and all of the Duchenne tubule volume, respectively.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eH\u003csub\u003e2\u003c/sub\u003eS production capability\u003c/h2\u003e \u003cp\u003eWine yeast produces trace amounts of H\u003csub\u003e2\u003c/sub\u003eS through sulfur metabolism, which has the odor of rotten eggs, garlic, or onions, negatively affecting the flavor profile of fruit wine. H\u003csub\u003e2\u003c/sub\u003eS can combine with bismuth to form a black bismuth sulfide precipitate, and the H\u003csub\u003e2\u003c/sub\u003eS production capability of the strains can be determined based on the color of the colonies on the selective medium BIGGY using bismuth as an indicator. Based on the levels of H\u003csub\u003e2\u003c/sub\u003eS production, the strains can be categorized into six levels: white, light brown, brown, light brown, brown, and black (Ying et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As shown in the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, strains HJ-2, HJ-3, and HJ-5 display white and light brown colors, classifying them as low H\u003csub\u003e2\u003c/sub\u003eS producers; strains HJ-6 and HJ-4 exhibit deep brown colors, categorizing them as mediumH\u003csub\u003e2\u003c/sub\u003eS producers; while strains HJ-1, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 show dark brown colors, indicating them as high H\u003csub\u003e2\u003c/sub\u003eS producers. Zheng et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) isolated four indigenous wine yeast strains from grape-growing regions to assess their H\u003csub\u003e2\u003c/sub\u003eS production capabilities and found that strains WJ1, Q12, and S21 were medium H\u003csub\u003e2\u003c/sub\u003eS producers, while strain S12 was a high H2S producer. Huang et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) isolated six non-wine yeast strains from naturally fermented goat milk fruit, where strains H1, H7, and H10 were white, strain H5 had light brown edges, and strains H9 and H12 were deep brown. This indicates that strains H1, H7, and H10 do not produce H\u003csub\u003e2\u003c/sub\u003eS, strain H5 produces low levels of H\u003csub\u003e2\u003c/sub\u003eS, and strains H9 and H12 produce high levels of H\u003csub\u003e2\u003c/sub\u003eS. Compared to the above studies, this demonstrates certain differences in the H\u003csub\u003e2\u003c/sub\u003eS production capabilities of yeast, further emphasizing the importance of yeast selection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEster production capability\u003c/h2\u003e \u003cp\u003eMicrobial secreted enzymes can catalyze the reaction between acids and alcohols to produce esters, which are further hydrolyzed into acids and alcohols, thereby enhancing the flavor of fruit wine (Yuan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). By inoculating yeast strains into a medium containing butyric glycerol ester and utilizing the esterases produced by the yeast to hydrolyze butyric glycerol ester, a transparent zone is formed that can be used to evaluate the ester production capability of the yeast strains (Ying et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, strains HJ-5, HJ-6, HJ-11, HJ-3, HJ-2, and HJ-8 exhibit significant transparent zones, indicating strong ester production capabilities; strains HJ-4, HJ-10, HJ-7, and HJ-12 have smaller transparent zones, indicating weaker ester production capabilities; while strains HJ-9 and HJ-1 show no obvious transparent zones, suggesting that these two yeast strains lack ester production capability. Long et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) assessed the ester production capabilities of five wine yeast strains from the fermentation liquor of Dian olive and found that the five strains exhibited varying ester production abilities, with strain LJM-10 demonstrating a strong ester production capacity. This indicates that there are certain differences in the ester production capabilities among different yeast strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMolecular biological identification\u003c/h2\u003e \u003cp\u003eThe 26S rDNA D1/D2 region within ribosomal DNA is commonly used for the molecular biological classification of yeast (Wang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). PCR amplification was performed on the 12 selected yeast strains, and as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the size of the 26S rDNA D1/D2 region in all 12 strains is approximately 600 bp, which matches the expected size. The sequencing results were compared with the NCBI database using BLAST (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and a phylogenetic tree was constructed (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 showed over 98% similarity to known strains of \u003cem\u003ePichia kudriavzevii\u003c/em\u003e AC1 (OP678979.1), \u003cem\u003ePichia kudriavzevii\u003c/em\u003e B-NC-13-OZ23 (KJ794697.1), \u003cem\u003ePichia kudriavzevii\u003c/em\u003e 11 (KR259307.1), \u003cem\u003ePichia kudriavzevii\u003c/em\u003e YZ4 (EU394711.1), \u003cem\u003ePichia kudriavzevii\u003c/em\u003e PEX-11 (MW990004.1), \u003cem\u003ePichia kudriavzevii\u003c/em\u003e 15 (KR259308.1), \u003cem\u003ePichia kudriavzevii\u003c/em\u003e L1 (EF126365.1), \u003cem\u003ePichia kudriavzevii\u003c/em\u003e feni48 (KM234455.1) and clustered with \u003cem\u003ePichia kudriavzevii\u003c/em\u003e CDA174Y2 (OQ568324.1), indicating that strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 belong to \u003cem\u003ePichia kudriavzevii\u003c/em\u003e. Strains HJ-2, HJ-3, HJ-4, and HJ-5 showed over 99% similarity to known strains of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e YC-D8 (OP644089.1), \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e QTX-D20 (OP644141.1), \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e QTX-D14 (OP644135.1), \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e YQY-F11 (OP644252.1)) and clustered with \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e UTAD97 (OQ305072.1), indicating that strains HJ-2, HJ-3, HJ-4, and HJ-5 belong to \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. It is widely recognized that strains with the same genotype tend to exhibit similar brewing characteristics (Bi et al. 2023). Therefore, one strain from \u003cem\u003ePichia kudriavzevii\u003c/em\u003e and one from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e with good fermentation performance, low H\u003csub\u003e2\u003c/sub\u003eS production, and high ester production (HJ-2, HJ-6) were selected for tolerance testing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequencing results of 26S rDNA D1/D2 sequence of five strain of yeast.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain Code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeries Length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHomologous Strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHomologous strain number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003edegree of similarity(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGenebanknumber\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e575 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePichia kudriavzevii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOP678979.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e585 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYC-D8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOP644089.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e589 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQTX-D20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOP644141.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e587 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eSaccharomyces cerevisiae\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQTX-D14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOP644135.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e585 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eSaccharomyces cerevisiae\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYQY-F11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOP644252.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e577 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ePichia kudriavzevii\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eB-NC\u0026minus;13-OZ23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKJ794697.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e582 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ePichia kudriavzevii\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKR259307.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e581 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ePichia kudriavzevii\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYZ4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEU394711.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e565 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ePichia kudriavzevii\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePEX\u0026minus;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW990004.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e582 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ePichia kudriavzevii\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKR259308.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e570 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ePichia kudriavzevii\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEF126365.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHJ\u0026minus;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e580 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ePichia kudriavzevii\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efeni48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKM234455.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTolerance of yeast strains\u003c/h2\u003e \u003cp\u003eThe pH of fruit wine typically ranges from 3.0 to 4.0, which necessitates the use of yeast with strong acid tolerance during the winemaking process; both excessively high and low pH levels can affect yeast activity (Ying et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, in this study, commercial yeast SY was used as a control strain to assess the pH tolerance of the selected strains HJ-2 and HJ-6. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (A), where it can be observed that as the environmental acidity increases, the growth condition of the strains gradually weakens. At a pH of 2.8, commercial yeast SY was able to grow normally, while the OD\u003csub\u003e600\u003c/sub\u003e nm values of strains HJ-2 and HJ-6 were below 0.5, approaching zero, indicating that the increased acidity significantly inhibited their growth. Conversely, as the acidity level decreased from pH 3.2 to pH 4.0, the growth rates of commercial yeast SY and strains HJ-2 and HJ-6 showed an upward trend, indicating their effective adaptability to these conditions. This finding is consistent with the results of Long et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which studied the pH tolerance of five yeast strains. Therefore, it can be concluded that strains HJ-2 and HJ-6 exhibit good tolerance to low pH environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEthanol is the primary metabolic product of yeast, and the accumulation of excessive ethanol in fruit wine fermentation can exert inhibitory and toxic effects on the yeast involved in the fermentation process, potentially leading to yeast cell death. The ethanol tolerance of yeast is closely related to its fermentation performance (Gan et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The ethanol tolerance of strains HJ-2, HJ-6, and commercial yeast SY was assessed, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (B). Within the ethanol concentration range of 3\u0026ndash;15%, the OD values of the yeast generally exhibited a downward trend as ethanol concentration increased. At an ethanol concentration of 3%, strains SY, HJ-2, and HJ-6 were able to grow normally. However, when the ethanol concentrations reached 6% and 9%, the growth of strain HJ-6 was inhibited, while strains SY and HJ-2 continued to grow normally. When the ethanol concentration increased to 12% and 15%, only strain HJ-2 grew normally, while the growth of strains SY and HJ-6 was inhibited. These results indicate that strain HJ-2 can tolerate ethanol concentrations of up to 15%, whereas the tolerance limits for strains SY and HJ-6 are 9% and 3%, respectively. Qin et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) found that the ethanol tolerance of the strain \u003cem\u003eHyphopichia burtonii\u003c/em\u003e Y9 was 9%, categorizing it as a strain with moderate ethanol tolerance. Additionally, Jiang et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that five aromatic yeast strains selected from high-temperature Daqu exhibited ethanol tolerances of 7%. Comparing these findings, it can be concluded that the selected strain HJ-2 demonstrates high ethanol tolerance, making it a suitable strain for the production of hawthorn fruit wine.\u003c/p\u003e \u003cp\u003eSugar is the primary substrate for ethanol production through fermentation and serves as the energy source for yeast. However, high sugar concentrations can inhibit yeast growth and reproduction, as high osmotic pressure may lead to water loss in yeast cells (Zhou et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The glucose and sucrose tolerance of strains SY, HJ-2, and HJ-6 were tested, with results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (C) and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (D). The commercial yeast SY exhibited minimal growth impact under various glucose and sucrose concentrations. Strain HJ-6 demonstrated tolerance when glucose concentration reached 300 g/L, while strain HJ-2 experienced varying degrees of inhibition as glucose concentration increased. When sucrose was used as the carbon source, strain HJ-2 maintained an OD\u003csub\u003e600\u003c/sub\u003e nm value greater than 1.0 at different sucrose concentrations, indicating strong tolerance to sucrose levels up to 400 g/L. These results highlight significant differences in sugar tolerance between strains HJ-2 and HJ-6 under different carbon sources. Zhao et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) isolated the strain F3 from naturally fermented mulberry juice, which exhibited the best tolerance, being able to withstand a maximum sugar concentration of 400 g/L. Mou et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) identified excellent strains B-6 and C-3 from Qingxiang Daqu that could tolerate glucose concentrations of 400 g/L. Compared to the aforementioned studies, the strains HJ-2 and HJ-6 selected in this research demonstrated good sugar tolerance.\u003c/p\u003e \u003cp\u003eIn wine production, the addition of SO\u003csub\u003e2\u003c/sub\u003e in appropriate amounts can inhibit harmful microorganisms and provide antioxidant and color-preserving effects. However, higher concentrations of SO\u003csub\u003e2\u003c/sub\u003e can adversely affect yeast strain growth, thereby prolonging fermentation time, and high osmotic pressure can also impact the metabolic products of yeast during alcoholic fermentation (Hu et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (E), strains HJ-2, HJ-6, and SY exhibited normal growth under different SO\u003csub\u003e2\u003c/sub\u003e concentrations, with no significant changes, indicating that these strains can tolerate SO\u003csub\u003e2\u003c/sub\u003e concentrations up to 360 mg/L. Gong et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) identified four yeast strains, YC-E8, QTX-D17, QTX-D7, and YQY-E18, from 168 local brewing yeast strains in Ningxia, which could tolerate 250 mg/L SO\u003csub\u003e2\u003c/sub\u003e. Zhang et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) isolated three excellent yeast strains, GL14, GP21, and GP24, from prickly pear, all of which could tolerate 300 mg/L SO\u003csub\u003e2\u003c/sub\u003e. These findings indicate that strains HJ-2 and HJ-6 exhibit good SO\u003csub\u003e2\u003c/sub\u003e tolerance, meeting the standards for high-quality yeast strains.\u003c/p\u003e \u003cp\u003eTemperature has a significant impact on yeast reproduction, with the optimal growth temperature for yeast being between 25\u0026deg;C and 30\u0026deg;C. Temperatures exceeding 35\u0026deg;C can lead to cell death; however, certain yeast strains can survive at temperatures above 40\u0026deg;C (Lai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, temperature tolerance tests were conducted on the selected strains, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (F), which displays the temperature tolerance of the three yeast strains. Under conditions of 4\u0026deg;C, 15\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, and 40\u0026deg;C, the OD values of the yeast strains initially increased and then decreased. At 4\u0026deg;C, the growth of commercial yeast SY, as well as strains HJ-2 and HJ-6, was inhibited. At 15\u0026deg;C and 25\u0026deg;C, strains SY and HJ-2 grew without inhibition, while strain HJ-6 was inhibited. At 30\u0026deg;C, all three yeast strains exhibited normal growth. At 40\u0026deg;C, strains SY and HJ-2 demonstrated better growth compared to strain HJ-6. These results indicate that the optimal temperature for strains SY, HJ-2, and HJ-6 is 30\u0026deg;C, with the ability to tolerate high temperatures of up to 40\u0026deg;C, which is consistent with the heat resistance findings for industrial yeast strains at 40\u0026deg;C.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, 12 yeast strains were isolated from the fermentation liquid of wild thorny fruits. Based on 26S rDNA D1/D2 region gene sequence analysis, strains HJ-1, HJ-6, HJ-7, HJ-8, HJ-9, HJ-10, HJ-11, and HJ-12 were identified as \u003cem\u003ePichia kudriavzevii\u003c/em\u003e, while strains HJ-2, HJ-3, HJ-4, and HJ-5 were identified as \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Two strains (HJ-2 and HJ-6) were selected for tolerance testing due to their good fermentation performance, high ester production, and low H\u003csub\u003e2\u003c/sub\u003eS production. Strain HJ-2 was able to grow normally under the following conditions: 40\u0026deg;C, 15% (v/v) ethanol, 360 mg/L SO\u003csub\u003e2\u003c/sub\u003e, pH 3.2, 400 g/L sucrose, and 250 g/L glucose. Strain HJ-6 grew normally under the conditions of 40\u0026deg;C, 3% (v/v) ethanol, 360 mg/L SO\u003csub\u003e2\u003c/sub\u003e, pH 3.2, 250 g/L sucrose, and 300 g/L glucose. Both strains HJ-2 and HJ-6 exhibited strong ester production capabilities and low H\u003csub\u003e2\u003c/sub\u003eS production. Gan et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) screened a strain of brewing yeast that could withstand pH 1.6, 18.2% ethanol concentration, 45\u0026deg;C temperature, and 250 mg/L SO\u003csub\u003e2\u003c/sub\u003e. Tan et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) isolated two brewing yeast strains from ten yeast strains that could grow and reproduce under conditions of 16% ethanol, 500 g/L glucose, 250 mg/L SO\u003csub\u003e2\u003c/sub\u003e, high temperature of 45\u0026deg;C, and low temperature of 5\u0026deg;C. Lei et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) isolated five non-brewing yeast strains with strong ester production capabilities from naturally fermented juice, among which strain Wa3 showed the best tolerance to high concentrations of alcohol, sugar, and SO\u003csub\u003e2\u003c/sub\u003e. Comparing with the above studies, strains HJ-2 and HJ-6 demonstrate excellent characteristics for fruit wine brewing. In summary, strains HJ-2 and HJ-6 are expected to provide yeast resources for fruit wine production and possess significant application potential in the thorny fruit wine industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were designed and conducted by L. Z., Data analysis was performed by J. Y. Y., Q. F. X., X. B., X. G., L. F. Z., W. L. L., H. H. W., and L. Z. The project was conceived and supervised by L. Z. The paper was written by L. Z. and J. C. with contributions from all authors, in accordance with the approval of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conficts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (Grant NO. 202301BA070001-081), Yunnan Fundamental Research Projects (Grant NO.\u0026nbsp;202301AU070012),\u0026nbsp;Special Basic Cooperative Research Innovation Programs of Qujing Science and Technology Bureau \u0026amp; Qujing Normal University (Grant NO. KJLH2023ZD06,\u0026nbsp;KJLH2022YB07),\u0026nbsp;National College Students Innovation and Entrepreneurship Training Program, China (Grant NO. G202310684015, G202310684002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo experiments involving human participants or animals were conducted by any of the authors in this study.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eBi JY, Lu LJ (2023) Screening of native \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and its application in navel orange wine brewing. China Brew \u003cstrong\u003e42\u003c/strong\u003e: 140-146.\u003c/li\u003e\n\u003cli\u003eChen K, Tan QQ (2021) Analysis of the nutritional components of \u003cem\u003ePyracantha\u003c/em\u003e fruits. 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Food Ferment Ind \u003cstrong\u003e46\u003c/strong\u003e: 79-84.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pyracantha fortuneana, Yeast, Isolation and Identification, Brewing characteristics","lastPublishedDoi":"10.21203/rs.3.rs-4842597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4842597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003ePyracantha fortuneana\u003c/em\u003e (\u003cem\u003eP. fortuneana\u003c/em\u003e) fruit, as a dual-purpose plant resource, is rich in nutrients; however, studies on the isolation and identification of yeast strains from \u003cem\u003eP. fortuneana\u003c/em\u003e fruit and their brewing characteristics are scarce. To screen for high-quality yeast strains specifically for \u003cem\u003eP. fortuneana\u003c/em\u003e wine from the fermentation liquid, this study employed traditional pure culture methods using WL nutrient agar for morphological identification of the isolated strains. Molecular biological identification of the yeasts was performed using molecular biology techniques. The brewing characteristics of the yeast strains were analyzed based on growth characteristics, sugar tolerance, alcohol tolerance, SO\u003csub\u003e2\u003c/sub\u003e tolerance, and acid tolerance, providing potential quality yeast resources for \u003cem\u003eP. fortuneana\u003c/em\u003e wine production. The results indicated that a total of 12 yeast strains were isolated and purified. After identification, they were classified as \u003cem\u003ePichia kudriavzevii\u003c/em\u003e and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Strain HJ-2 was able to grow normally under the conditions of 40\u0026deg;C, 15% alcohol by volume, 360 mg/L SO\u003csub\u003e2\u003c/sub\u003e mass concentration, pH 3.2, 400 g/L sucrose mass concentration, and 250 g/L glucose mass concentration. Strain HJ-6 exhibited normal growth under conditions of 40\u0026deg;C, 3% alcohol by volume, 360 mg/L SO\u003csub\u003e2\u003c/sub\u003e mass concentration, pH 3.2, 250 g/L sucrose mass concentration, and 300 g/L glucose mass concentration. Both strains HJ-2 and HJ-6 demonstrated strong ester production capability and low hydrogen sulfide production. Overall, strains HJ-2 and HJ-6 show potential for application in the industrial production of \u003cem\u003eP. fortuneana\u003c/em\u003e wine.\u003c/p\u003e","manuscriptTitle":"Isolation, identification, and brewing characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-29 16:37:41","doi":"10.21203/rs.3.rs-4842597/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-16T07:13:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-14T08:16:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-11T17:52:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197517115958912788697012009597656418009","date":"2024-08-10T14:53:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152172561192476213706172361624736797298","date":"2024-08-08T07:39:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157862317773822900680938034076766742272","date":"2024-08-07T18:27:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76340350835843874351744328317040144991","date":"2024-08-06T08:16:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75824306210619475298209871727697855739","date":"2024-08-05T13:07:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60220513978408628307936013428542307539","date":"2024-08-05T08:06:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-05T07:56:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-03T00:15:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-02T02:46:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2024-08-01T13:38:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c241ad1c-ac24-4958-a01e-7fb8c461928c","owner":[],"postedDate":"August 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:11:56+00:00","versionOfRecord":{"articleIdentity":"rs-4842597","link":"https://doi.org/10.1007/s00203-024-04164-4","journal":{"identity":"archives-of-microbiology","isVorOnly":false,"title":"Archives of Microbiology"},"publishedOn":"2024-10-18 15:58:08","publishedOnDateReadable":"October 18th, 2024"},"versionCreatedAt":"2024-08-29 16:37:41","video":"","vorDoi":"10.1007/s00203-024-04164-4","vorDoiUrl":"https://doi.org/10.1007/s00203-024-04164-4","workflowStages":[]},"version":"v1","identity":"rs-4842597","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4842597","identity":"rs-4842597","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}